Project title: Function and mechanism of LGR4 and LGR5 in modulation of Wnt signaling Abstract LGR4 and LGR5 (leucine-rich repeat containing G protein-coupled receptor 4 and 5) are two related receptors of the rhodopsin-like 7TM receptor superfamily with critical roles in development and oncogenesis. Heterozygous mutation of LGR4 is associated with several human diseases while complete knockout of LGR4 in mice leads to embryonic lethality with hypoplasia and defective tubulogenesis in several organs. LGR5 is now the most recognized marker of adult stem cells in the gastrointestinal tract and other epithelial tissues, although it is not essential for the self-renewal of LGR5-positive stem cells. We and others discovered that LGR4/5 function as receptors of R-spondins (RSPOs) to potentiate Wnt signaling. RSPOs are a group of four related secreted proteins (RSPO1-4) with distinct roles in organ development and survival of stem cells. Recently, recurrent gene fusions of RSPO2 and RSPO3 were identified in a subset of colon and prostate cancers. The discovery of RSPOs as ligand of LGR4/5 provides a molecular basis for stem cell-specific effect of the Wnt signaling and for the pleiotropic functions that LGR4/5 and R-spondins have in development, stem cell survival, and oncogenesis. Despite containing a 7TM domain typical of the rhodopsin family of GPCRs, simulation of LGR4/5 by RSPOs does not lead to activation of heterotrimeric G proteins or ?- arrestin. Instead, RSPO-LGR4 potentiates Wnt signaling via two pathways, inhibition of ubiquitin ligases that degrade Wnt receptors and recruitment of the scaffold protein IQGAP1 that coordinate Wnt signaling. However, the exact roles and signaling mechanisms of the RSPO- LGR4/5 system in the regulation of stem cell fitness and Wnt signaling remain poorly understood. In this proposal, we will carry out three aims to dissect how RSPO, LGR4/5, and IQGAP1 interact to regulate their downstream mediators, to identify and characterize the function and mechanism of LGR5 in the regulation of cell-cell adhesion and stem cell fitness, and to delineate the mechanisms of RSPO-LGR4 activation in potentiation of Wnt signaling. The results and conclusions will provide important insights not only into the governing principles of Wnt signaling and stem cell survival but also the discovery and development of novel therapeutics in regenerative medicine and cancer treatment.